Cluster of Differentiation 43 Deficiency in Leukocytes Leads to Reduced Atherosclerosis—Brief ReportSignificance
Objective—The aim of this study was to investigate the role of cluster of differentiation 43 (CD43), an integral membrane glycoprotein with both proadhesive and antiadhesive activities, in atherosclerosis.
Approach and Results—Low-density lipoprotein receptor–deficient mice were lethally irradiated and reconstituted with either bone marrow from CD43−/− mice or from wild-type controls. We found that mice lacking the CD43 on their leukocytes had significantly less severe atherosclerosis and that, contrary to our expectation, macrophage infiltration into the vessel wall was not affected by the lack of CD43 in the leukocytes. However, we found that CD43 mediates cholesterol homeostasis in macrophages by facilitating cholesterol efflux. This resulted in a significant reduction in storage of cholesterol in the aorta of mice lacking CD43 in the leukocytes.
Conclusions—CD43 may be an important mediator of macrophage lipid homeostasis, thereby affecting macrophage foam cell formation and ultimately atherosclerotic plaque development.
Macrophages are present at every stage of atherosclerosis from fatty streak lesion to fibrofatty plaque.1 Migration of leukocytes into the vessel wall is a well-orchestrated series of events involving molecules, such as selectins, integrins, and chemokines produced locally at the arterial sites. Equally important are the receptors on the surfaces of the leukocytes that are necessary for the interaction with their ligands on the endothelium.2
One such ligand is cluster of differentiation 43 (CD43), an integral membrane glycoprotein that is commonly found on the surface of all hematopoietic cells. Based on its structural characteristics of the extracellular domain, CD43 can potentially have both proadhesive properties resulting from the extracellular domain being extensively O-glycosylated3 or antiadhesive with extensive negatively charged sialic acid residues on its extracellular domain.4 The abundant expression and far-reaching protrusion from the cell surface3 make CD43 a likely candidate to be the first point of contact with other cell surfaces. Adhesion would be facilitated in cells that express 1 of the known counter–receptors for CD43, intercellular adhesion molecule 1, galectin 1, and major histocompatibility complex-1.5–7 Interactions with other cells that do not express a CD43 counter–receptor would likely result in cell repulsion because of steric hindrance and electrostatic repulsion by the negatively charged molecules.
Because of the abundance of CD43 expression on leukocytes and its intriguing proadhesive and antiadhesive properties, we conducted studies to determine whether leukocyte CD43 plays a role in atherosclerosis.
Materials and Methods
Materials and Methods are available in the online-only Data Supplement.
To assess whether CD43 plays a role in atherogenesis, low-density lipoprotein receptor–deficient mice were lethally irradiated and reconstituted with bone marrow isolated from either CD43−/− mice (CD43−/−BMT [bone marrow transplantation]) or control mice (CD43+/+BMT) and fed a high-fat diet for additional 16 weeks to induce atherosclerosis. After 4 weeks of post-BMT fluorescence-activated cell sorting analysis of peripheral blood revealed that most of the CD11b+ myeloid cells in the CD43−/−BMT mice did not express CD43, whereas the control mice showed abundant expression of CD43 (Figure I in the online-only Data Supplement).
During the 16 weeks on the high-fat diet, the plasma cholesterol and triglyceride levels were virtually identical between the CD43+/+BMT and CD43−/−BMT mice at all time points (Table I in the online-only Data Supplement). After 16 weeks, the mice were euthanized, and the aortic valve in the heart was sectioned and stained with Oil red O to quantify the lesion areas (Figure 1A). The lesions from the CD43−/−BMT mice were appreciably smaller and less advanced compared with lesions from the control CD43+/+BMT mice (571 481±90 110 versus 772 980±93 543 μm2; P<0.0001). The aortas from these mice were longitudinally opened and cut into 2 pieces immediately below the superior mesenteric artery. The top portion was used for quantifying the percent lesion area (Figure 1B). The aortic lesions were much less extensive in the CD43−/−BMT mice compared with those in the controls (15.1±2.5% versus 9.0±2.2%; P<0.0001). Both measures indicated that leukocyte-specific deficiency of CD43 protected the mice from atherosclerosis.
Quantitative analysis of lesion morphology by immunohistochemistry indicated that macrophage accumulation (Figure IIA in the online-only Data Supplement) in the plaques was not different in the lesions of CD43−/−BMT mice compared with those in the CD43+/+BMT mice. With n=8 for each group, the MOMA2-stained areas as a percentage of total plaque area were 34.7±5.9 for the CD43+/+BMT mice and 30.3±5.3 for the CD43−/−BMT mice (mean±SEM). Furthermore, CD43−/− and CD43+/+ macrophages displayed similar propensity to migrate in an macrophage colony–stimulating factor–mediated transwell migration assay (Figure IIB in the online-only Data Supplement).
The lower portion of the aorta was used to measure the cholesterol content by extracting the lipids and separating them via thin layer chromatography. There was a dramatic reduction in the cholesteryl ester content of the aortas from CD43−/−BMT mice compared with controls (46±12 versus 174±26 mg/mL; P<0.001). Free cholesterol was also lower in the CD43−/−BMT mice (33±5 versus 61±9 mg/mL; P<0.01).
Because our thin layer chromatography results showed far less cholesterol deposited in the lesions of the CD43−/−BMT mice, we performed both cholesterol uptake and efflux experiments to assess whether CD43 participates in either of these processes using primary macrophages isolated from CD43−/− and CD43+/+ mice. The results showed that whereas cholesterol uptake was not affected by CD43 (Figure III in the online-only Data Supplement), cholesterol efflux was significantly enhanced in macrophages lacking CD43 (Figure 2B). This suggests that the CD43 on macrophages acts to inhibit cholesterol efflux from foam cells and explains the reduced cholesterol deposits in the aortas of CD43−/−BMT mice. However, expression levels of genes that are known to be involved in both cholesterol uptake (cluster of differentiation 36 and scavenger receptor A) and efflux (ATP-binding cassette [ABC] A1 and ABCG1), as well as inflammatory cytokine, such as interleukin-1β, were not different between the 2 groups of mice (Figure IV in the online-only Data Supplement). These results indicate that CD43 participates in lipid homeostasis in macrophages without affecting the cadre of genes known to regulate these processes.
Although CD43 is expressed abundantly on macrophages, the role that CD43 has on its function has not been studied carefully. Using an ex vivo assay, McEvoy et al8 demonstrated that an anti-CD43 antibody can inhibit the adhesion of macrophages to the aortic endothelium isolated from rabbits fed a cholesterol-rich diet and therefore more prone to atherogenesis. Although these results are interesting, it is not clear whether the results obtained could be extrapolated to the in vivo model. Indeed, using our in vivo model, it was clear that disruption of CD43 on macrophages did not affect the accumulation of these cells in the plaque despite significantly less atherosclerosis in the lesions of CD43−/−BMT mice. This suggests that CD43 deficiency on leukocytes had other effects that led to limited progression of the disease.
Our finding that CD43 mediates cholesterol metabolism is novel and unexpected. We found that the accumulation of cholesteryl ester was reduced by 74% and free cholesterol by 45% in CD43−/−BMT mice. Furthermore, assessing the capabilities of macrophages to metabolize cholesterol, we found that cholesterol efflux was markedly enhanced in CD43−/− macrophages compared with controls. These results indicate that CD43 normally inhibits efflux of cholesterol from macrophages without directly affecting the expression of efflux-enhancing membrane proteins, ABCA1 and ABCG1, or the scavenger receptors, cluster of differentiation 36 and scavenger receptor A. It is possible that CD43 may interact in some way with these transporters on the cell membrane to prevent them from shuttling cholesterol out of the cell. It is also possible that CD43 may block the interaction of ABCA1 or ABCG1 with potential cholesterol acceptors, such as HDL and apolipoprotein A-I. Our findings suggest that inhibiting CD43 may constitute an interesting therapeutic strategy to limit the development of atherosclerosis.
We thank thank Hongwei Wang for technical assistance. This work was performed within the Russian Government Program of Competitive Growth of Kazan Federal University.
Sources of Funding
This work was supported by National Institutes of Health (NIH) grants R01HL075677 and R01HL81863 and Hawaii Community Foundation grant 10ADVC-47037 to W.A. Boisvert. Core facilities were supported by NIH grants P20GM103516, P20RR016453, G12RR003061, and G12MD007601.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.114.304513/-/DC1.
- Nonstandard Abbreviation and Acronym
- bone marrow transplantation
- Received December 5, 2012.
- Accepted December 3, 2014.
- © 2014 American Heart Association, Inc.
- Hernandez JD,
- Nguyen JT,
- He J,
- Wang W,
- Ardman B,
- Green JM,
- Fukuda M,
- Baum LG
- Stöckl J,
- Majdic O,
- Kohl P,
- Pickl WF,
- Menzel JE,
- Knapp W
- McEvoy LM,
- Jutila MA,
- Tsao PS,
- Cooke JP,
- Butcher EC
Cluster of differentiation 43 (CD43) is an integral membrane glycoprotein that is expressed on all leukocytes but its function has not been clearly elucidated. To assess the role of CD43 in atherogenesis, atherosclerosis-prone low-density lipoprotein receptor–deficient mice were lethally irradiated and transplanted with bone marrow from either CD43−/− mice or from the control CD43+/+ mice. The extent of atherosclerosis was less severe in low-density lipoprotein receptor–deficient mice that received CD43−/− marrow than that in those that were transplanted with bone marrow from control mice. Further work revealed that CD43 hinders with the process of transporting cholesterol out of lipid-filled macrophages. This study identifies CD43 as a potential target in trying to combat atherosclerosis. It would be relatively easy to inhibit this protein to enhance the reverse cholesterol transport by the macrophages with the ultimate goal of slowing down the development of atherosclerosis.